Sepiapterin reductase deficiency (SRD), an extremely rare but treatable neurotransmitter disease, is an enzyme defect in the final step of tetrahydrobiopterin (BH4) synthesis.1 Unlike other forms of BH4-deficient dopa-responsive dystonia, SRD uniquely does not manifest hyperphenylalaninemia and thus slips through detection by newborn screening. Owing to its variable presenting features and need for a sensitive method of CSF analysis, diagnosis of SRD may be compromised in mild phenotypes.2
We describe a novel splice site variant leading to leaky splicing control of the SPR gene. Our observation adds evidence to the notion that leaky splicing may take part in SRD heterogeneity and evokes the image of an iceberg beneath the water: patients at the milder end of the spectrum escaping recognition.
Case report
An 8-month-old girl presented with postural limb dystonia that worsened in the evening. Brain imaging, EEG, routine blood, urine, and CSF testing were nondiagnostic. Recognition of her episodic oculogyric crises and convergence spasms prompted us to analyze her CSF for pterins and biogenic amines. CSF homovanillic acid (132 nmol/L) and 5-hydroxyindoleacetic acid (11.5 nmol/L) were decreased (normal range: 295–932 nmol/L and 114–336 nmol/L, respectively). The CSF BH4 level, analyzed by the method described by Fukushima and Nixon,3 was below the detection limit, whereas total biopterin (27.06 nmol/L) and neopterin (22.06 nmol/L) levels were within the normal range, suggesting that most of the patient's total biopterin was a sum of biopterin and dihydrobiopterin. Findings were suggestive of monoamine neurotransmitter disease due to BH4 deficiency. l-dopa/carbidopa therapy completely suppressed her dystonia and resulted in near-normal psychomotor development.
Genetic analysis established the diagnosis of SRD by identifying compound heterozygous variants in the SPR gene (NM_003124.4): c.512G>A and c.304+1_+12del. The former is a novel missense variant, absent in the Exome Aggregation Consortium (ExAC) and gnomAD databases, estimated to substitute a well-conserved cysteine for tyrosine, and predicted as damaging according to in silico analyses. The latter, also absent in the ExAC and gnomAD databases, destroys the 5′ splice donor site in intron 1, rendering the gene prone to aberrant splicing (figure, A).
(A) The patient was compound heterozygous for an exonic c.512G>A and an intronic c.304+1_+12del (SPR, NM_003124.4) (B and C) The destroyed splice site and retention of intron 1 resulted in a larger size 899-bp band (B, arrow) specific to the patient. Because allelic origin was identifiable based on the c.512G>A variant, sequencing the normally spliced 319-bp product (B, arrowhead) showed significant wild-type splicing from the allele carrying c.304+1_+12del (C). Primers were designed as depicted, and sequences are available upon request.
Next, splicing analysis was performed, using blood cell transcripts extracted from the patient and a healthy control. Primers were designed to flank intron 1 and exon 2 of the SPR gene and to specifically amplify the RNA sequences (figure B). Reverse transcription-PCR–based splicing analysis not only confirmed aberrant splicing causing intron retention (figure B, arrow) but also discovered evidence for leaky splicing control related to c.304+1_+12del. Because the allelic origin was identifiable based on the presence or absence of c.512G>A, directly sequencing the shorter 319-bp amplicon (figure B, arrowhead) showed significant wild-type splicing from the allele carrying c.304+1_+12del (figure C).
Discussion
Leaky splicing control contributes to phenotypic variation by affecting disease onset and/or severity. The extent of leaky wild-type transcription determines, for example, residual acid alpha-glucosidase activity in Pompe disease and relates to a specific-form of adult-onset disease.4 As for SRD, others have reported the possibility of leaky splicing causing intrafamilial heterogeneity.5 In the report, however, splicing was assessed indirectly using the minigene system. Our report proves by directly analyzing patient RNA that leaky splice site variants indeed underlie SRD. Phenotypic variability owing to such leaky splicing control may further expand the SRD spectrum.
Study funding
Supported by grants from Morinaga Hoshikai and AMED (JP18ek0109280 and JP18ek0109301).
Disclosure
Y. Nakagama has received government research support from JSPS Kakenhi and has received foundation/society research support from Morinaga Hoshikai. K. Hamanaka and M. Mimaki report no disclosures. H. Shintaku has received government research support from the AMED. S. Miyatake has received foundation/society research support from the Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics, JSPS KAKENHI, and The Ichiro Kanehara Foundation for the Promotion of Medical Science & Medical Care. N. Matsumoto has served on the editorial boards of Clinical Genetics, Journal of Human Genetics, and American Journal of Medical Genetics and has received foundation/society research support from the AMED, JSPS KAKENHI, and Takeda Science Foundation. K. Hirohata reports no disclosures. R. Inuzuka has served on the editorial board of Journal of Pediatric Cardiology and Cardiac Surgery and has received government research support from JSPS KAKENHI. A. Oka has received funding for travel or speaker honoraria from Otsuka Pharmaceutical, UCB Japan, Nobelpharma, Janssen Pharmaceutical, Eisai, BioMarin Pharmaceutical, Novartis Pharma K.K., GE Healthcare Japan, Teijin Pharma Limited, Shionogi & Co., Shire Japan, Bayer Yakuhin, and SRL Inc.; has served on the editorial board of Pediatrics International; has commercial research support from Maruho Co, Pfizer Japan, Astellas Pharma, Novartis Pharma K.K., Chugai Pharmaceutical Co., and Eli Lilly Japan K.K.; and has received government funding from The Ministry of Health Labour and Welfare Japan. Disclosures available: Neurology.org/NG.
Appendix Author contributions
Footnotes
↵* These authors contributed equally to this work.
Funding information and disclosures are provided at the end of the article. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG.
The Article Processing Charge was funded by the authors.
Patient consent: Written consent for publication was obtained from the patient's family.
- Received October 26, 2018.
- Accepted in final form February 8, 2019.
- Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.
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